Evaluating the Immunogenicity of Protein Drugs by Applying In Vitro

The immune system has evolved to become highly specialized in recognizing and responding to pathogens and foreign molecules. Specifically, the function of HLA class II is to ensure that a sufficient sample of peptides derived from foreign molecules is presented to T cells. This leads to an important concern in human drug development as the possible immunogenicity of biopharmaceuticals, especially those intended for chronic administration, can lead to reduced efficacy and an undesired safety profile for biological therapeutics. As part of this review, we will highlight the molecular basis of antigen presentation as a key step in the induction of T cell responses, emphasizing the events associated with peptide binding to polymorphic and polygenic HLA class II molecules. We will further review methodologies that predict HLA class II binding peptides and candidate epitopes. We will focus on tools provided by the Immune Epitope Database and Analysis Resource, discussing the basic features of different prediction methods, the objective evaluation of prediction quality, and general guidelines for practical use of these tools. Finally the use, advantages, and limitations of the methodology will be demonstrated in a review of two previous studies investigating the immunogenicity of erythropoietin and timothy grass pollen

Insights into HLA-Restricted T Cell Responses in a Novel Mouse Model of Dengue Virus Infection Point toward New Implications for Vaccine Design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for human leukocyte antigens (HLA) are widely used to model human immune responses and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR−/− mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR−/− mice with HLA A*0201, A*0101, A*1101, B*0702 and DRB1*0101 transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR−/− strains tested. In contrast only 8 of these elicited responses in the corresponding IFN-α/βR+/+ mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV exposed human donors, and a dominance of HLA B*0702 restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we herein describe a novel murine model, which allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design

Complement C3d Conjugation to Anthrax Protective Antigen Promotes a Rapid, Sustained, and Protective Antibody Response

B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax™) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure

Three dimensional lithospheric structure of the western continental margin of India constrained from gravity modelling: implication for tectonic evolution

This paper describes a 3-D lithospheric density model of the Western Continental Margin of India (WCMI) based on forward modelling of gravity data derived from satellite altimetry over the ocean and surface measurements on the Indian peninsula. The model covers the north-eastern Arabian Sea and the western part of the Indian Peninsula and incorporates constraints from a wide variety of geophysical and geological information. Salient features of the density model include: (1) the Moho depth varying from 13 km below the oceanic crust to 46 km below the continental interior; (2) the lithosphere–asthenosphere boundary (LAB) located at depths between 70 km in the southwestern corner (under oceanic crust) and about 165 km below the continental region; (3) thickening of the crust under the Chagos–Laccadive and Laxmi Ridges and (4) a revised definition of the continent–ocean boundary. The 3-D density structure of the region enables us to propose an evolutionary model of the WCMI that revisits earlier views of passive rifting. The first stage of continental-scale rifting of Madagascar from India at about 90 Ma is marked by relatively small amounts of magmatism. A second episode of rifting and large-scale magmatism was possibly initiated around 70 Ma with the opening of the Gop Rift. Subsequently at around 68 Ma, the drifting away of the Seychelles and formation of the Laxmi Ridge was a consequence of the down-faulting of the northern margin. During this second episode of rifting, the northern part of the WCMI witnessed massive volcanism attributed to interaction with the Reunion hotspot at around 65 Ma. Subsequent stretching of the transitional crust between about 65 and 62 Ma formed the Laxmi Basin, the southward extension of the failed Gop Rift. As the interaction between plume and lithosphere continued, the Chagos–Laccadive Ridge was emplaced on the edge of the nascent oceanic crust/rifted continental margin in the south as the Indian Plate was moving northwards

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Regulation of B cell receptor signaling : B cell activation through complement and the role of protein tyrosine phosphatase Shp2

My thesis is focused upon mechanisms regulating B cell receptor (BCR) activation and regulation. There are two branches of the immune system, the innate and adaptive. Proper cross-talk between the innate and adaptive immune system is crucial in initiating proper and effective immune responses and preventing inappropriate immune reactions such as autoimmunity and lymphoma. Arguably the most crucial link between B cell function and the innate immune system is through the complement receptor CD21 (Cr2). CD21 is the receptor for the complement breakdown product C3d. Cross-linking of antigen with C3d has been shown to augment antigen specific antibody titers far above normal immune responses. In chapter 1, I have shown that conjugation of protective antigen (PA) of anthrax with C3d produces 3-fold greater antibody titers as compared to PA in alum - the vaccine currently being developed for human administration. These antigen-specific antibodies are produced very rapidly during the primary response, are of all IgG isotypes, and are sustained long after primary immunization. Finally, I see that C3d may augment this response by enhancing the efficiency of germinal center formation and plasma cell differentiation. In chapter 2, I focus upon the mechanism of CD21 function on B cells and follicular dendritic cells (FDCs). By immunizing WT and CD21-/- mice, I found that CD21 is important in initiating proper B cell function during the primary immune response but had little role in eliciting secondary or memory immune responses. I further examine CD21 function by examining the mechanisms by which CD21 functions on the surface of B cells. By using deconvolution microscopy and immunoprecipitation, I lend support to the sequestration model of CD21 function. This model implicates CD21 is an inhibitory receptor on B cells which actively sequesters CD19, the cardinal member of the BCR co-receptor complex. Finally, in chapter 3, I look downstream of surface receptor regulation of BCR function and look at the role of the protein tyrosine phosphatase, Shp2, in B cell development, signaling and function. By using Cre/lox-p technology, I create a B cell specific deletion of Shp2 and show that Shp2 may be an important regulator of marginal zone versus follicular B cell development and is a crucial regulator in the GC reactio

Regulation of B cell receptor signaling : B cell activation through complement and the role of protein tyrosine phosphatase Shp2

My thesis is focused upon mechanisms regulating B cell receptor (BCR) activation and regulation. There are two branches of the immune system, the innate and adaptive. Proper cross-talk between the innate and adaptive immune system is crucial in initiating proper and effective immune responses and preventing inappropriate immune reactions such as autoimmunity and lymphoma. Arguably the most crucial link between B cell function and the innate immune system is through the complement receptor CD21 (Cr2). CD21 is the receptor for the complement breakdown product C3d. Cross-linking of antigen with C3d has been shown to augment antigen specific antibody titers far above normal immune responses. In chapter 1, I have shown that conjugation of protective antigen (PA) of anthrax with C3d produces 3-fold greater antibody titers as compared to PA in alum - the vaccine currently being developed for human administration. These antigen-specific antibodies are produced very rapidly during the primary response, are of all IgG isotypes, and are sustained long after primary immunization. Finally, I see that C3d may augment this response by enhancing the efficiency of germinal center formation and plasma cell differentiation. In chapter 2, I focus upon the mechanism of CD21 function on B cells and follicular dendritic cells (FDCs). By immunizing WT and CD21-/- mice, I found that CD21 is important in initiating proper B cell function during the primary immune response but had little role in eliciting secondary or memory immune responses. I further examine CD21 function by examining the mechanisms by which CD21 functions on the surface of B cells. By using deconvolution microscopy and immunoprecipitation, I lend support to the sequestration model of CD21 function. This model implicates CD21 is an inhibitory receptor on B cells which actively sequesters CD19, the cardinal member of the BCR co-receptor complex. Finally, in chapter 3, I look downstream of surface receptor regulation of BCR function and look at the role of the protein tyrosine phosphatase, Shp2, in B cell development, signaling and function. By using Cre/lox-p technology, I create a B cell specific deletion of Shp2 and show that Shp2 may be an important regulator of marginal zone versus follicular B cell development and is a crucial regulator in the GC reactio

Immunization with PA-Av-C3d₃/IFA augments production of PA- and Domain 4-specific antibodies relative to alum.

<p>Groups of A/J (top), BALB/c (middle), and C3H/HeJ (bottom) mice were immunized with PA-Av/IFA (○), PA-Av-Alum (□), or PA-Av-C3d₃/IFA (▵). (A) Serum PA- and (B) Domain 4-specific IgG was measured by ELISA (Abs 405 nm). All mice were boosted at day 42 (↑) with PA in saline. Error bars indicate SEM (n = 5). (C) Endpoint ELISAs were performed on day 14 serum samples from PA-Av/IFA, PA-Av-Alum, and PA-Av-C3d₃/IFA immunized mice. Endpoint titers shown are 2-fold above background. Error bars represent SEM (n = 5, *p≤0.05, ** p≤0.01, *** p≤0.005).</p